This invention relates to the use of a substantially pure, water soluble azo dye i.e. Basic Blue 54 for the cytological preparation of a fixed biopsy specimen derived from human blood, bone marrow, lymph nodes and other specimen of hematopoietic origin. The use of a water soluble azo dye (identified in the Colour Index as Basic Blue 54 in accordance with this invention, is an advance over the prior art (Romanowsky and Malachowski) wherein a mixture of dyes were used for staining biopsy specimens. The stained cells of hematopoietic origin have excellent Colours stability and are remarkably clear with respect to cellular detail and brilliance of cell structure.
Blood contains a variety of cells. The most numerous cells are the erythrocytes, or red blood cells, which carry out the exchange of oxygen and carbon dioxide between the lungs and the body tissues. The minor population of cells are the leucocytes, or white blood cells, which control the immuno response system of the body and defend the body against infecting organisms and foreign agents both in the tissues and the bloodstream. The leucocyte population in blood is further defined by a number of subclasses which play distinct roles in the immune response. For example, the relative number cells in various subclasses of lymphocytes (about 20% of the leucocyte population) is likely to change in various disease states. Identification of cells of the various subclasses provides an indication of the relative well being of the patient.
The staining of biological cells and tissues with dyes, in order to differentiate one from another or to render them more easily observable under a microscope or other means is known in the art. By such means it is possible to differentiate, for example, among the five types of peripheral blood leucocytes: the neutrophils, eosinophils, basophils, lymphocytes, and monocytes; the difference between cancerous and normal cells; and the difference between mature and immature cells. This differentiation enables the cytologist to diagnose certain diseases.
More specifically, Ehrlich introduced the use of dyes to effect or enhance cell differentiation in human biopsy specimens particularly blood cells. Ehrilich's dyes were superceded, however, by the use of mixtures of dyes identified as Romanowsky dyes which have been modified to include mixtures such as methylene blue, modified methylene blues, eosins, azures and methylene violet. These mixtures of dyes have been generally classified as panoptic stains because of the wide range and broad spectra of hues and chroma produced when reacted with a fixed biological specimen such as human blood. As early as 1891, Romanowsky and Malachowski developed mixtures of polychromed methylene blue, azure and methylene violet. Other contributors include Unna (1891), Nocht (1898), Jenner (1899), Leishman (1901), Wright (1902), May-Grunwald and Giemsa (1902), MacNeal (1906) and Lillie (1943).
The diagnosis of hematological disorders has been achieved, for the most part, by enumeration and identification of formed elements of the peripheral blood and bone marrow. The basis of hematological diagnosis includes the use of light microscopic examination of a panoptically stained specimen of blood cells, lymph node cells, or bone marrow cells. This type of examination provides information sufficient to make a diagnosis which parallels the developments of panoptic light microscopy. Cytochemical stains have been developed to identify cell types more precisely than was possible by using panoptic stains. Cytochemistry represents biochemistry on a microscopic and submicroscopic level. When applied to cells and tissue, cytochemical stains identify enzymes, substrates and organelles. As biochemical probes, cytochemical stains often provide valuable insights regarding aberrations of cellular metabolism. Moreover, cytochemical stains can be selective for one cell type compared to another and therefore such stains have been used in various diagnostic processes especially in making distinctions between various cytological types of acute leukemias and preleukemic disorders.
With the development of the synthetic organic dyes, various investigators experimented with supravital stains by adding these dyes to freshly obtained samples of blood or suspension of cells. It was rapidly ascertained that some of the cells were stained with one or more of the dyes whereas other cells were not. Subsequent to the development of supravital staining of blood cells, Ehrlich found the need for a more stable, permanent preparation of blood cells that could be examined under the microscope. Recognizing the difficulties in cell identification in viewing suspensions of unstained cells, Ehrlich devised a stain composed of orange G, acid fuschin and methyl green. On the basis of differential coloration of leukocytes with a mixture of dyes, Ehrlich identified and named most of the blood leukocytes known today. Ehrlich's contribution was remarkable in that by using a plurality of dyes he was able to detect the difference in colors that were distinctive for various cell types. For example, those cells whose granules showed affinity for eosin were called eosinophils. Recognizing that some cells stained differently than the color of the dye in solution, the term metachromasia was popularized and applied to the granules of mast cells. At present, staining techniques form the basis of modern morphological hematology and the nomenclature of various cell types.
However, early in the history of the morphologic and cytochemical diagnosis of blood disorders, it was found that examination of only panoptically stained specimens of blood or bone marrow was not sufficient to make a diagnosis. While some investigators were popularizing the supravital stains, others were describing cytochemical stains for blood cells that could be used on dried, fixed preparations of blood or bone marrow. It became apparent that there were some blood cells that had peroxidase activity while other cells did not. The peroxidase test was the first stain that reliably distinguished between granulocytic cells which contain activity of peroxidase and lymphoid cells which did not contain peroxidase activity. The peroxidase stains with chromophoric modifications and increased use in immunology remain one of the most useful stains in the cytochemistry of blood cells and is the basis for one of the current automated leukocyte differential counting instruments.
As a staining technique, however, cytochemistry has limitations with respect to age of sample, type of fixative, pH, presence or absence of heavy metal cations, deterioration of the substrate, time and temperature of the staining reaction, etc. These are all variables that affect the cytochemical stain. In addition, any impurities in the organic dyestuff as well as variability in the composition of the dye stuff causes alterations in the staining reaction. However, by using cytochemical stains, it is possible to identify the presence or absence of substances in one cell type contrasted to another or any increase or decrease in the quantity of such substance in those cell types. Quantitatively, these differences assume a diagnostic importance when they reflect differences in one cell type compared to another, and in normal cells compared to abnormal or pathological blood cells.
In several instances, specific diseases have cytochemical profiles that complement the traditional microscopic examination of panoptically stained preparations. There are a variety of hematologic disorders wherein cytochemical tests have diagnostic value. Complementing the conventional light microscopy of panoptically stained specimens of blood or bone marrow, cytochemical stains have improved the precision of hematological diagnosis with the recognition that these stains can reveal properties that are distinctive for one cell type compared to another. Cytochemical stains have found increased application in the study of blood, lymph node and bone marrow specimens. For the most part, these stains detect increased or decreased amounts of an enzyme or a metabolite that reflect the pathophysiological condition of a disordered cell. While the exact mechanism or chemistry responsible for the production of the cytochemical abnormalities are unknown, many of these abnormalities are sufficiently distinctive to make them useful diagnostically. As a diagnostic tool for cellular hematology, cytochemistry represents a rapid and inexpensive method to distinguish one cell type from another on the basis of characteristic properties. With advances in dye chemistry one can anticipate further improvements in the cytochemistry of blood cells and the precision of hematological diagnosis in the future. Discussions of cytochemical stains can be found in Cytochemical Stains for Blood and Bone Marrow Cells and Cystobiology of Leukemias and Lymphomias, by L. Kass, M.D., Raven Press, New York, New York, Pages 161-177, 1983, and Lawrence Kass, M.D. Leukemia Cytology and Cytochemistry, published by J.B. Lippincott, Philadelphia, 1982.
In 1898, Ehrlich and Lazarus described a large transitional cell in the peripheral blood. This cell had abundant cytoplasm, and convoluted nucleus. Most likely, this cell was a monocyte. In 1912, Schilling named a similar appearing cell "monozyten". In 1913, Reschad and Schilling described the first case of monocytic leukemia. In panoptically stained preparations of peripheral blood or bone marrow, monocytes are large mononuclear cells with indented or convoluted nuclei, coarse appearing nuclear chromatin strands, and abundant cytoplasm that sometimes contains a few azurophilic granules. In living monocytes, pseudopodia can be seen at the cytoplasmic borders, and the cell displays random movement on a glass coverslip.
For more precise identification of the monocyte, the cytochemical reaction for nonspecific esterase activity has been used. With either alpha naphthyl acetate or preferably alpha naphthyl butyrate as the substrate and fast blue BBN or hexazotized pararosanalin as the coupler, activity of nonspecific esterase is intense in monocytes and in macrophages which is the end stage cell of the monocytic series. Furthermore, nonspecific esterase activity in monocytes is sensitive to inhibition by sodium fluoride, and can be obliterated if sodium fluoride is added to the incubation mixture. Monoclonal antibodies may be used also to identify monocytes, but these antibodies may show cross-reactivity with other cells thereby reducing the specificity for monocytes.
In bone marrow, the promyelocyte is the precursor cell of the monocyte. The monocyte is known as the scavenger cell of the blood. It actively phagocytizes or engulfs foreign particles, such as bacteria, and destroys them. Increased numbers of monocytes are found in the blood in a variety of disorders, such as acute and chronic infections, tropical disorders such as schistosomiasis, syphilis, trypanosomiasis, tuberculosis, myelodysplastic disorders (preleukemic conditions), and monocytic leukemia. Recently, it has been shown that the HIV virus responsible for AIDS can reside unharmed within the cytoplasm of monocytes, and thereby be disseminated throughout the body by these cells. Increasingly, the monocyte has become important in understanding the immune process, even though its role in immunity is complex and critical.